Extended Data Fig. 4: Characterization of Ras-LOCKR-PL.

a–c, Representative western blots of CIAR-PM-293 cells treated with 500 μM biotin with or without 250 nM A115 (labeled ‘A’) for 16 h. (a) Negative controls (Myc-tagged Key or Flag-tagged Cage only), positive control (TurboID-CRaf), and Ras-LOCKR-PL tested designs (Key+Cage) were expressed, and treated with DMSO or with A115. Cell lysates were subjected to a streptavidin pulldown (PD) and the amount of Ras eluted from the streptavidin PD over the amount of Ras from whole cell lysate (WCL) was quantified (n = 5 biologically independent experiments per condition). (b) Optimized Ras-LOCKR-PL co-expressed with various HA-tagged Ras mutants/effectors or Rap effector (CalDAG-GEFI) (n = 3 biologically independent experiments per condition). (c) Localized Ras-LOCKR-PLs were tested for A115-induced biotinylation in CIAR-PM-293 cells (representative of 3 biologically independent experiments). d, Representative epifluorescence images from 3 biologically independent experiments of EKAR4²⁹ localized to PM (N-terminus of Lyn)²² or Golgi (N-terminus of eNOS)²³ transfected into 293T cells, which were immunostained for their respective localization markers. e, 293T cells transfected with localized EKAR4 and either with or without Ras-LOCKR-PL also localized to same region. Normalized FRET ratio changes over time of these cells stimulated with 100 ng/mL EGF (n = 12 cells per condition). Solid lines indicate representative average timecourse with error bars representing standard error mean (s.e.m.). Bar graphs represent mean ± s.e.m. **p < 0.01, *p < 0.05, unpaired two-tailed Student’s t-test. Scale bar = 10 μm.